A CD25×TIGIT bispecific antibody induces anti-tumor activity through selective intratumoral Treg cell depletion

Xin Wei; Linlin Zhao; Fang Yang; Yajing Yang; Huixiang Zhang; Kaixin Du; Xinxin Tian; Ruihua Fan; Guangxu Si; Kailun Wang; Yulu Li; Zhizhong Wei; Miaomiao He; Jianhua Sui

doi:10.1016/j.ymthe.2024.09.010

. 2024 Sep 7;32(11):4075–4094. doi: 10.1016/j.ymthe.2024.09.010

A CD25×TIGIT bispecific antibody induces anti-tumor activity through selective intratumoral Treg cell depletion

Xin Wei ^1,², Linlin Zhao ², Fang Yang ², Yajing Yang ², Huixiang Zhang ², Kaixin Du ², Xinxin Tian ², Ruihua Fan ², Guangxu Si ², Kailun Wang ², Yulu Li ², Zhizhong Wei ², Miaomiao He ², Jianhua Sui ^2,^3,^∗

PMCID: PMC11573620 PMID: 39245938

Abstract

Intratumoral regulatory T cells (Tregs) express high levels of CD25 and TIGIT, which are also recognized as markers of effector T cell (Teff) activation. Targeting these molecules each alone with monoclonal antibodies (mAbs) poses a risk of concurrently depleting both Teffs and peripheral Tregs, thereby compromising the effectiveness and selectivity of intratumoral Treg depletion. Here, leveraging the increased abundance of CD25⁺ TIGIT⁺ double-positive Tregs in the solid tumor microenvironment (but not in peripheral tissues), we explore the feasibility of using a CD25×TIGIT bispecific antibody (bsAb) to selectively deplete intratumoral Tregs. We initially constructed a bsAb co-targeting mouse CD25 and TIGIT, NSWm7210, and found that NSWm7210 conferred enhanced intratumoral Treg depletion, Teff activation, and tumor suppression as compared to the parental monotherapies in mouse models. We subsequently constructed a bsAb co-targeting human CD25 and TIGIT (NSWh7216), which preferentially eliminated CD25⁺ TIGIT⁺ double-positive cells over single-positive cells in vitro. NSWh7216 exhibited enhanced anti-tumor activity without toxicity of peripheral Tregs in CD25 humanized mice compared to the parental monotherapies. Our study illustrates the use of CD25×TIGIT bsAbs as effective agents against solid tumors based on selective depletion of intratumoral Tregs.

Keywords: intratumoral regulatory T cells, Treg depletion, bispecific antibody, bsAb, CD25, TIGIT

Graphical abstract

Sui and colleagues developed bispecific antibodies (bsAbs) targeting CD25 and TIGIT by leveraging the abundance of immune-suppressive CD25⁺ TIGIT⁺ double-positive regulatory T cells (Tregs) in tumor microenvironment. Experiments with mouse models showed that the bsAbs safely enhances intratumoral Tregs depletion and tumor suppression and outperforms the parental monoclonal antibodies.

Introduction

Regulatory T cells (Tregs) are indispensable components of the normal immune system,¹ and enrichment of Tregs in tumors has been reported to be associated with poor prognosis in cancer patients.²^,³^,⁴ Given the known immunosuppressive activities of Tregs, depleting these cells has been considered as a potential strategy to unleash the function of effector cells in the tumor microenvironment.⁵ Antibody (Ab)-based Treg depletion has achieved tumor control in preclinical models: for instance, Abs targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4) can deplete Tregs and suppress tumor growth via an Fc-dependent mechanism.⁶ However, the clinical efficacy of Ab-based Treg depletion is limited by low efficiency of intratumoral Treg depletion,⁷ unwanted depletion of Teffs,⁸ and adverse events related to the interference of the peripheral Tregs.⁹ These challenges underscore that enhancing both the specificity and efficiency of intratumoral Treg depletion agents could facilitate development of more efficacious cancer therapies.

Bispecific Abs (bsAbs) are considered a promising strategy for selectively targeting particular cells expressing two independent antigens, and dual antigen-expressing cells can be targeted specifically by adjusting the bsAbs' affinity.¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶ To obtain a bsAb targeting intratumoral Tregs, it is essential to choose appropriate phenotypic markers that feature relatively high expression on Tregs and low expression on Teffs. Two categories of Treg surface receptors have been reported to mediate two types of suppressive mechanisms of Tregs: (1) high interleukin (IL)-2 receptor subunit-α (IL2RA, CD25) expression on Tregs, which can mediate competitive consumption of IL-2 with consequent apoptosis of Teffs¹⁷; and (2) immune co-inhibitory molecules, such as CTLA-4, lymphocyte activation 3 (LAG-3), and T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM)domains (TIGIT), which can mediate immunosuppression by suppressing T cell functions.¹⁸^,¹⁹

CD25 is expressed constitutively on Tregs, absent on naive Teffs, and upregulated on activated Teffs during tumor development.²⁰^,²¹ Previous anti-CD25 mAbs tested in the clinic (daclizumab and basiliximab) and in preclinical models can block IL-2 binding with CD25.²¹^,²²^,²³ Recently, multiple research groups have generated anti-CD25 mAbs that do not block IL-2 binding with CD25; these can preserve IL-2 signaling activation in Teffs, providing enhanced anti-tumor activity compared to anti-CD25 mAbs that inhibit the binding of IL-2 with CD25.²⁴^,²⁵^,²⁶ However, anti-CD25 mAbs also significantly deplete the peripheral Tregs,²⁴ and its clinical efficacy and safety still require validation (NCT04158583).

TIGIT has been conceptualized as a promising inhibitory immune checkpoint; during tumor development, it is expressed on activated natural killer (NK) cells and T cells, including CD4⁺ T cells, CD8⁺ T cells, and Tregs.²⁷^,²⁸^,²⁹^,³⁰ Quantification of TIGIT density in tumor-infiltrating lymphocytes (TILs) from patients with several solid tumor types revealed intratumoral Tregs as the population expressing the highest TIGIT receptor density.³¹ The interaction of TIGIT with its cognate ligand CD155 is known to induce inhibitory signals in T cells, NK cells, and APCs.²⁸^,²⁹^,³⁰^,³²^,³³ Notably, Tregs with TIGIT expression have been shown to be more immunosuppressive than TIGIT-deficient Tregs,³²^,³⁴^,³⁵ and preclinical research has revealed both ligand-binding blockade and elimination of Tregs as dual mechanisms for anti-TIGIT mAbs with functional Fc.³¹^,³⁶^,³⁷ Moreover, anti-TIGIT mAbs were demonstrated to be well tolerated as a monotherapy and in combination with other mAbs (such as anti-programmed cell death protein 1 [PD-1]/programmed death-ligand 1 [PD-L1] mAbs).³⁸^,³⁹^,⁴⁰

We thus speculated that co-targeting CD25 and TIGIT with a bsAb should selectively reduce intratumoral Tregs, ideally while sparing activated Teffs and periphery Tregs. Here, we generated bsAbs that co-engage with CD25 and TIGIT. Based on the increased abundance of CD25⁺ TIGIT⁺ double-positive Tregs in the tumor microenvironment (but not in peripheral tissues), we initially designed and constructed an anti-mouse CD25×TIGIT bsAb, NSWm7210. NSWm7210 outperforms monotherapies in terms of Treg depletion, Teff activation, and tumor suppression in mouse tumor models. We also constructed an anti-human CD25×TIGIT bsAb, NSWh7216, with improved anti-tumor activity compared to its parental mAbs, while not reducing the proportion of peripheral Tregs in tumor mouse models. Thus, our study shows that a bsAb targeting CD25 and TIGIT can improve the selectivity and efficiency of Treg depletion and achieve efficient anti-tumor activity against established solid tumors.

Results

CD25 and TIGIT are co-expressed in both murine and human intratumoral Tregs

CD25 and TIGIT have been reported as highly expressed on intratumoral Tregs, respectively.²¹^,³¹ To explore the feasibility of using a bsAb co-engaging with CD25 and TIGIT to specifically target intratumoral Tregs, we first examined the co-expression of CD25 and TIGIT on murine Tregs and Teffs. Using flow cytometry, we profiled CD25 and TIGIT expression on individual T cell subsets (CD4⁺ FoxP3⁺ Treg, CD4⁺ FoxP3⁻, and CD8⁺ Teffs) inside tumors, spleens, and peripheral blood (Figures 1A and S1). When comparing the proportions of CD25⁺ TIGIT⁺ double-positive cells (CD25⁺ TIGIT⁺), CD25⁺ single-positive cells (CD25⁺ TIGIT⁻), and TIGIT⁺ single-positive cells (CD25⁻ TIGIT⁺) in tumors from the MC38 colon carcinoma mouse model, we observed that, among intratumoral Tregs, approximately 83.0% were CD25⁺ TIGIT⁺ double-positive cells, while this double-positive population represented only 30% of intratumoral CD4⁺ FoxP3⁻ Teffs and 15% of intratumoral CD8⁺ Teffs, respectively. In contrast, in spleens and peripheral blood, about 60% of Tregs are CD25⁺ TIGIT⁻, while only 11% of Tregs in blood and 27% of Tregs in spleens are CD25⁺ TIGIT⁺ (Figure 1A). In other mouse models, including E.G7 lymphoma and CT26 colon carcinoma, the CD25 and TIGIT co-expression pattern closely resembles that observed in the MC38 mouse model (Figure 1A). Thus, CD25⁺ TIGIT⁺ Tregs are enriched in the tumor microenvironment of MC38, EG.7, and CT26 mouse models, supporting the potential use of a bsAb simultaneously engaging CD25 and TIGIT to specifically target intratumoral Tregs.

Generation of NSWm7210, an anti-mouse CD25×TIGIT bsAb

(A) CD25 and TIGIT expression on Foxp3⁺ CD4⁺, Foxp3^– CD4⁺, and CD8⁺ T cells derived from tumor tissues, blood, and spleens from three syngeneic tumor mouse models. MC38 and E.G7 tumors were established in C57BL/6 mice. CT26 tumors were established in BALB/c mice. The ratios of CD25⁺ TIGIT⁻, CD25⁻ TIGIT⁺, and CD25⁺ TIGIT⁺ cells among CD4⁺Foxp3⁺, CD4⁺Foxp3⁻, and CD8⁺ T cells—isolated from tumor tissues, blood, and spleens—were analyzed when tumors reached approximately 300–500 mm³, n = 3 per group. The data are presented as the mean ± SEM, and are representative of two independent experiments. (B) Schematic diagram of NSWm7210 construction. Dark colors denote heavy-chain domains and light colors denote light-chain domains. For each bispecific Ab, hetero-dimerization of the two heavy chains was achieved using the knobs-into-holes method; the heavy chain with a bulge (left) is the knob chain and the other one (right) is the hole chain. The double black lines represent a disulfide bond between the two heavy chains. (C) NSWm7210 binding concurrently with mCD25 and mTIGIT antigens as measured by an SPR assay (depicted in the schematic). The blue and magenta circles respectively denote mCD25 and mTIGIT. R1, binding response of NSWm7210 to the immobilized mCD25; R2, binding response of mTIGIT to NSWm7210. (D) Evaluation of IL-2 blocking ability of anti-mCD25 mAbs and NSWm7210. CD3⁺ T cells were isolated from C57BL/6 mouse splenocytes. A total of 500,000 cells were plated and allowed to rest for 1 h at 37°C. Abs were added at 10 μg/mL, with incubation for 30 min at 37°C, after which cells were stimulated with human (h) IL-2 (100 U/mL) for 30 min at 37°C. Cells were stained for phosphorylated STAT5 (pSTAT5), and the pSTAT5 signal in Tregs and Teffs was recorded. The representative histograms show changes of pSTAT5 signal density observed in Tregs after incubation with the indicated Abs. (E) The binding selectivity of the NSWm7210 for mCD25⁺ mTIGIT⁺ CHO cells. Pre-stained mCD25⁺ mTIGIT⁺ CHO cells and mCD25⁺ CHO cells were mixed with non-stained mTIGIT⁺ CHO cells at a ratio of 1:1:1. Cells were incubated with serially diluted NSWm7210 prior to staining with phycoerythrin (PE)-conjugated anti-human(h) IgG secondary Ab. Binding of NSWm7210 with the indicated populations of CHO cells was assessed by flow cytometry based on the PE fluorescence intensity for each cell population. Data shown are representative data of three independent experiments. See also Figures S1–S3, and Tables S1 and S2.

To test if CD25⁺ TIGIT⁺ Tregs are present in human tumors, we used a pan-cancer T cell atlas⁴¹ and obtained single-cell RNA sequencing (scRNA-seq) data from 21 tumor types to analyze CD25 and TIGIT expression in CD4⁺ TILs and CD8⁺ TILs. CD25 and TIGIT were co-expressed at higher levels on Tregs, especially on two subsets of Tregs (TNFRSF9⁺ Tregs and OAS1⁺ Tregs) compared to other CD4⁺ subsets (Figures S2A–S2C). Notably, TNFRSF9⁺ Tregs have been reported as the most abundant population of CD4⁺ TILs, while their frequencies among CD4⁺ T cell populations in blood and normal tissues are significantly lower in comparison.⁴¹ We also found that CD25 was expressed at a low level on CD8⁺ TILs, while TIGIT was expressed at a higher level on four distinct exhausted CD8⁺ T (Tex) cells compared to other CD8⁺ subsets (Figures S2E–S2G). As lung cancer, colorectal cancer, and liver cancer are among the top five cancer types globally in terms of both incidence and mortality,⁴² we obtained scRNA-seq data for patient cohorts from these three tumor types⁴¹ for further analyses. In the three representative tumor types, the CD25 and TIGIT co-expression patterns in CD4⁺ TILs (Figure S2D) and CD8⁺ TILs (Figure S2H) closely resemble those observed in our pan-cancer data analysis.

Design and characterization of a bispecific antibody targeting mouse CD25 and TIGIT

Considering the anti-CD25-depleting mAbs that do not interfere with IL-2 binding to CD25 can induce strong effector responses and anti-tumor activity,²⁴^,²⁵^,²⁶ we initially adopted a non-IL-2-blocking anti-mouse CD25 mAb (7D4)⁴³ along with an anti-TIGIT mAb (CS19) to construct a bsAb co-targeting mouse CD25 (mCD25) and mouse TIGIT (mTIGIT), which we termed NSWm7210 (Figure 1B). Surface plasmon resonance (SPR) analysis showed that 7D4 has a picomolar range affinity for mCD25 (Figure S3A). CS19 was obtained by screening our phage display naive Ab library (single-chain fragment of variable domain [scFv])⁴⁴; it has nanomolar-range affinities for both mTIGIT and human TIGIT (hTIGIT) (Figure S3A). Competition ELISA assay showed that CS19 effectively blocked the interaction between TIGIT and its receptor CD155 (Figure S3B). NSWm7210 was generated by engineering 7D4 and CS19 mAbs with knobs-into-holes and Cross-Mab technologies.⁴⁵^,⁴⁶ All bsAbs used in this study were purified via protein A affinity chromatography from supernatants after transient transfection of HEK293F cells. SPR supported the dual binding specificity of NSWm7210 for the mCD25 and mTIGIT antigens (Figure 1C).

Given the essential functions of IL-2 in the survival and function of Teffs,⁴⁷ we evaluated the IL-2-blocking activities of 7D4 mAb and NSWm7210 by quantification of phosphorylated STAT5 (pSTAT5). Following IL-2 stimulation, mouse CD3⁺ splenocyte-derived Foxp3⁺ Tregs exhibited an approximately 67.1% increase in pSTAT5 signal intensity (Figure 1D), whereas no increase in the pSTAT5 signal was detected in CD8⁺ or CD4⁺ Teffs (Figure S3C). Pre-treatment of mouse CD3⁺ splenocytes with an anti-mCD25 mAb (PC61) with partial IL-2 blocking ability induced a nearly 36.5% reduction in the pSTAT5 signal in Foxp3⁺ Tregs, whereas pre-treatment with an anti-IL-2 neutralizing mAb (NARA1) ablated the STAT5 signal (Figure 1D); in contrast, pre-treatment with 7D4 mAb and NSWm7210 did not affect the pSTAT5 signal of Foxp3⁺ Tregs (Figure 1D). These results establish that NSWm7210 does not block IL-2 signaling after binding to CD25, suggesting that NSWm7210 will not interfere with IL-2-mediated Teff function.

To examine whether NSWm7210 is selective for binding to CD25⁺ TIGIT⁺ cells, we first constructed Chinese hamster ovary (CHO) cell lines expressing either mCD25, mTIGIT alone, or a combination of mCD25 and mTIGIT (Figure S3D). To mimic the natural expression level ratios of CD25 and TIGIT on murine Tregs (Table S1), we selected a stable mCD25⁺ mTIGIT⁺ double-positive CHO cell line with the expression of mCD25 in approximately 4-fold excess of mTIGIT by fluorescence-activated cell sorting (FACS) (Table S2). The expression levels of CD25 and TIGIT on both single-positive cells and double-positive cells are at similar levels (Table S2).

Next, the binding selectivity of NSWm7210 for double-positive cells was assessed by pre-staining the three types of CHO cells with distinct tracer dyes, mixing them in equal ratios, and then incubating them with serially diluted NSWm7210, 7D4 mAb, or CS19 mAb. Flow cytometry analysis showed that NSWm7210 had an at least 6-fold stronger affinity for mCD25⁺ mTIGIT⁺ double-positive CHO (half-maximal effective concentration [EC₅₀] = 0.09 μg/mL) than for mCD25⁺ CHO cells (EC₅₀ = 0.59 μg/mL), and NSWm7210 also exhibited at least a 3-fold greater maximal binding capacity for double-positive CHO cells compared to mCD25⁺ CHO cells at the highest concentration tested. Additionally, NSWm7210 showed much stronger binding activity for double-positive CHO cells than for mTIGIT⁺ CHO cells. This was shown by its relatively low EC₅₀ (0.09 μg/mL) for binding to the double-positive CHO cells, but its binding with mTIGIT⁺ CHO cells was weak and the EC₅₀ could not be measured accurately; a maximal binding capacity for double-positive CHO cells was at least 6-fold higher than that for mTIGIT⁺ CHO cells. In contrast, 7D4 and CS19 had no selectivity for mCD25⁺ mTIGIT⁺ CHO cells (Figure 1E). Thus, we have constructed NSWm7210, an anti-mouse CD25×TIGIT bsAb, that is selective for mCD25⁺ mTIGIT⁺ double-positive cells.

NSWm7210 suppresses the growth of established tumors in an Fc-dependent manner

After establishing NSWm7210’s selectivity in binding with CD25⁺ TIGIT⁺ double-positive cells, we examined whether NSWm7210 may confer improved anti-tumor activity over the parental mAbs in vivo. We compared the anti-tumor activity of NSWm7210 with 7D4 mAb, CS19 mAb, and a combination therapy comprising 7D4 mAb and CS19 mAb in four syngeneic tumor models (MC38, E.G7, CT26, and MCA205) in wild-type (WT) mice. Once tumors reached 80–200 mm³ in size, the tumor-bearing mice were divided and treated with Abs twice a week with a dosage of 10 mg/kg. Compared to vehicle controls, both NSWm7210 and the combination therapy significantly inhibited tumor growth and prolonged the survival of mice in the MC38, E.G7, and CT26 tumor models (Figures 2A–2C). In the MC38 and E.G7 tumor models, for the tested monotherapies, only 7D4 significantly inhibited tumor growth, but its anti-tumor activity was significantly weaker than NSWm7210 (Figures 2A and 2B). In the CT26 tumor model, there was no significant difference in anti-tumor activities among NSWm7210, 7D4 mAb, and CS19 mAb (Figure 2C). However, the MCA205 tumor-bearing mice were resistant to the NSWm7210 monotherapy (Figure S4A), suggesting a potential need to combine NSWm7210 with other immunotherapies to overcome this resistance.

NSWm7210 suppresses the growth of established tumors

(A) Anti-tumor activity of NSWm7210 and the parental mAbs in the MC38 tumor model. C57BL/6 mice were inoculated subcutaneously (s.c.) with 300,000 MC38 tumor cells. When tumors reached approximately 80–200 mm³, the mice were divided into five groups with similar mean tumor volumes and treated i.p. with Abs (10 mg/kg) on day 8 post tumor cell inoculation. For the combination therapy, each mAb was administered at a dose of 5 mg/kg. Growth curves (left) and survival curves (right) of MC38 tumors are show, n = 5–6 per group. (B) Anti-tumor activity of NSWm7210 and the parental mAbs in the E.G7 tumor model. C57BL/6 mice were inoculated s.c. with 500,000 E.G7 tumor cells. When tumors reached approximately 80–200 mm³, mice were divided into five groups with similar mean tumor volumes and treated i.p. with Abs (10 mg/kg) on day 6 post tumor cell inoculation. For the combination therapy, each mAb was administered at a dose of 5 mg/kg. Growth curves (left) and survival curves (right) of EG.7 tumors are shown, n = 5–6 per group. (C) Anti-tumor activity of NSWm7210 and the parental mAbs in the CT26 tumor model. BALB/c mice were inoculated s.c. with 200,000 CT26 tumor cells. When tumors reached approximately 100–400 mm³, mice were divided into five groups with similar mean tumor volumes and were treated i.p. with Abs (10 mg/kg) on day 10 post tumor cell inoculation. For the combination therapy, each mAb was administered at a dose of 5 mg/kg. Growth curves (left) and survival curves (right) of CT26 tumors are shown, n = 5–6 per group. Data shown are representatives of at least two independent experiments. The time points for Ab treatment are marked by arrows. Tumor volumes were measured by caliper, and the data are shown as the mean ± SEM; statistical significance was determined using two-way ANOVA. See also Figures S4 and S5.

Given that anti-PD-1 therapy has become a standard treatment option for many cancer patients,⁴⁸ we further investigate whether NSWm7210 can synergize with anti-mouse PD-1 (mPD-1) to improve anti-tumor activity against the MCA205 tumor mouse model. We compared the anti-tumor efficacy of anti-mPD-1 monotherapy, a combination therapy comprising NSWm7210 with anti-mPD-1, and a combination therapy comprising 7D4 with anti-mPD-1 (Figure S4). Once tumors reached 80–200 mm³, the tumor-bearing mice were divided and treated with a single dose of NSWm7210 or 7D4 (5 mg/kg), followed by anti-mPD-1 (4 mg/kg) treatment on day 9 and day 12 post tumor cell inoculation (Figure S4B). Compared to the vehicle control, we found that anti-mPD-1 monotherapy did not effectively inhibit MCA205 tumor growth; in contrast, the combination therapy of NSWm7210 with anti-mPD-1 significantly improved the anti-tumor activity and prolonged the survival of MCA205 tumor-bearing mice (Figures S4C and S4D). These results indicate that NSWm7210 can synergize with another immune checkpoint inhibitor (such as anti-PD-1 Abs) to improve anti-tumor activity.

To evaluate the impact of the NSWm7210 and anti-mPD-1 combination therapy on activated intratumoral CD8⁺ T cells, we compared the percentage of activated (TIGIT⁺ PD-1⁺ CD39⁺) CD8⁺ T cells following the combination therapy and the anti-mPD-1 monotherapy using flow cytometry (Figure S4E). Compared to the anti-mPD-1 monotherapy, mice receiving the NSWm7210 and anti-mPD-1 combination therapy exhibited a 58% increase in activated (TIGIT⁺ PD-1⁺ CD39⁺) CD8⁺ T cells (Figure S4F). This suggests that the combination therapy can result in an increase in the subpopulation of activated CD8⁺ T cells, rather than causing depletion.

To test whether the anti-tumor activity of NSWm7210 depends on Fc-mediated effector functions, we first assessed if a functional Fc capable of binding to FcγRs is required for its in vivo anti-tumor activity. By comparing the anti-tumor efficacy of NSWm7210 containing a functional Fc to NSWm7210 bearing a human Fc DANG variant (DANG: with mutations D265A and N297G in its Fc region to eliminate binding activity to all classes of FcγRs⁴⁹) in the MC38 tumor mouse model, we found that, compared to the vehicle control, NSWm7210-DANG significantly suppressed MC38 tumor growth; however, its anti-tumor effect was considerably diminished (Figure S5). Thus, the anti-tumor activity of NSWm7210 depends on Fc-FcγRs interactions.

NSWm7210 promotes intratumoral Treg depletion and effector cell activation in the tumor microenvironment

To test whether the improved anti-tumor activity of NSWm7210 depends on stronger intratumoral Treg depletion, we next used flow cytometry to assess changes in T cell subsets in the tumor microenvironment after Ab treatment in the MC38 tumor model. Briefly, when MC38 tumor reached 300–500 mm³ in size on day 14, mice were treated with a single dose (5 mg/kg) of Abs, and 3 days later mice were sacrificed for intratumoral Treg analysis. The detected changes in both the percentage and absolute number of Foxp3⁺ Tregs after treatment showed that NSWm7210 induced significantly stronger intratumoral Treg depletion compared to either of the monotherapies or to the combination therapy (Figures S6, 3A, and 3B).

To investigate the durability of Treg depletion after NSWm7210 treatment cessation, we conducted flow cytometry analyses to compare the percentage of intratumoral Tregs on days 3, 8, and 14 after NSWm7210 and parental mAbs treatment. Briefly, a single dose of 5 mg/kg of NSWm7210 treatment led to depletion of intratumoral Tregs for approximately 14 days, which is slightly longer than the intratumoral Treg depletion observed with 7D4 (about 12 days). Notably, compared to 7D4, NSWm7210 was able to maintain intratumoral Tregs at a 1.2- to 2.4-fold lower level (Figure 3C).

After confirming NSWm7210’s capacity for intratumoral Treg depletion, we examined its potential influence on effector cells. Based on a flow cytometry analysis, we found that NSWm7210 significantly increased the CD8⁺/Treg, CD4⁺ Teff/Treg, and NK/Treg ratios compared to the other treatments 3 days after Ab treatment (Figure 3D), which indicated that NSWm7210 has minor influence on effector cells while effectively depleting intratumoral Tregs. Additionally, the sustained impacts of NSWm7210 treatment on effector cells was directly evaluated on days 3, 8, and 14 after a single dose of Ab (5 mg/kg) treatment (Figure S7). Briefly, 3 days after NSWm7210 treatment, there were no decreases in the proportions of CD3⁺ T, CD8⁺ T, NK, or CD4⁺ Teffs compared to the isotype control; on the eighth day after NSWm7210 treatment, there were significant increases in the proportions of CD8⁺ T and NK cells. By the 14^th day after NSWm7210 treatment, the proportions of CD8⁺ and NK cells returned to the same levels as the isotype control group (Figure S7).

We then evaluated the effects of NSWm7210 treatment on proinflammatory cytokine production in CD8⁺ T and NK cells in the tumor microenvironment to assess the potential of NSWm7210 to induce a proinflammatory microenvironment. Compared to the isotype mAb control, NSWm7210 treatment induced 2.7-fold higher production of interferon (INF)-γ in CD8⁺ T cells, 2.9-fold higher production of INF-γ in NK cells, and 1.5-fold higher production of perforin in NK cells; similar levels of induction of these cytokines (INF-γ and perforin) in CD8⁺ or NK cells were also observed for the combination of two mAbs, while the monotherapies did not induce any increase in cytokine productions in CD8⁺ T or NK cells (Figure 3E). These findings support that the anti-tumor activity of NSWm7210 can be attributed to both intratumoral Treg depletion and effector cell activation in the tumor microenvironment.

Assessment of NSWm7210’s safety profile

The depletion of normal tissue-derived Tregs can cause adverse effects such as autoimmunity and allergies.⁵⁰^,⁵¹ We assessed the possible impact of the NSWm7210 treatment on peripheral Tregs by assessing the Treg depletion in spleens and peripheral blood after a single-dose Ab treatment. In both blood and spleens, NSWm7210 appeared to reduce the proportion of Tregs to a degree similar to that of the 7D4 mAb and the combination therapy (Figure 3F). Taken together, our findings suggest that NSWm7210, at the cost of reducing the similar number of peripheral Tregs, is more efficient than the 7D4 mAb monotherapy and the combination treatment for intratumoral Treg elimination.

We also investigated potential off-target effects of NSWm7210 treatment on intestinal tissues. To this end, we first analyzed CD25 and TIGIT expression in intestinal Tregs. We isolated lamina propria cells from the cecum of WT C57BL/6 mice (see section “materials and methods”). Using flow cytometry, we profiled the expression of CD25 and TIGIT in three individual T cell subsets (CD4⁺ FoxP3⁺ Treg, CD4⁺ FoxP3⁻ Teffs, and CD8⁺ Teffs) of the intestine. We observed that, among lamina propria-derived Tregs, approximately 50% were CD25⁺ TIGIT⁻, while around 20% were CD25⁺ TIGIT⁺ double positive (Figure S8A). The co-expression pattern of CD25 and TIGIT in intestinal Tregs was more similar to what was observed in peripheral and splenic Tregs than in intratumoral Tregs (Figure 1A).

We then used flow cytometry to assess potential changes in intestinal T cell subsets of WT C57BL/6 mice following four doses of NSWm7210 treatment (10 mg/kg, twice per week). Five days after the final treatment, the mice were sacrificed for analysis of intestinal Tregs, T helper (Th) 1, and Th17 cells. Flow cytometry analysis showed that NSWm7210 induced a 27% decrease in the percentage of Tregs (Figure S8B); there were no changes in the percentages of inflammatory Th1 or Th17 cells (Figures S8C and S8D). These results indicate that, although NSWm7210 disturbed the Treg population in the intestine, it did not lead to activation of inflammatory CD4⁺ T cell subsets. We also performed hematoxylin and eosin (H&E) staining to assess histological changes in the intestines. There were no histological changes or inflammatory T cell infiltration in the intestines in the NSWm7210 or control groups (Figure S8E).

Immunogenicity refers to the ability of therapeutic Abs to induce unwanted immune responses, and can be in part characterized by the development of anti-drug Abs (ADAs).⁵² ADAs can, in theory, neutralize the therapeutic Ab and/or accelerate its clearance, thereby potentially altering its pharmacokinetics (PK) profile and safety.⁵³ To investigate the potential immunogenicity of NSWm7210, WT C57BL/6 mice (n = 5) were treated intraperitoneally (i.p.) with NSWm7210 (10 mg/kg) twice per week for five doses; blood serum samples were collected at baseline (day 0), as well as on days 3, 7, 14, 21, and 28 prior to each NSWm7210 administration. The ADAs in serum samples were examined using ELISA. The result showed that all animals receiving NSWm7210 developed ADAs by day 7 of the treatment regimen (Figures S9A and S9B).

Additionally, the PK properties of Abs (7D4, CS19, and NSWm7210) were characterized following a single dose of Abs (10 mg/kg) in WT C57BL/6 mice. Comparisons of the PK profiles of NSWm7210, 7D4, and CS19 showed that NSWm7210 had a shorter serum half-life (t_1/2 ∼51.3 h) compared to CS19 (t_1/2 ∼124.8 h) and 7D4 (t_1/2∼ 103.0 h) (Figure S9C); NSWm7210 reached its peak serum concentration relatively fast (T_max ∼ 3 h, C_max ∼ 124.4 ng/mL), within 3 h, while CS19 (T_max ∼ 24 h, C_max ∼ 138.9 ng/mL) and 7D4 (T_max ∼ 24 h, C_max ∼ 114.8 ng/mL) took ∼24 h to reach their peak concentrations (Figure S9C). These results suggest that NSWm7210 induces immunogenicity in mice, which may be attributed to its human immunoglobulin (Ig) G isotype, but this may not informatively represent its immunogenicity profile in humans.

To evaluate the potential immune-related adverse events after NSWm7210 treatment, we also assessed changes in peripheral T lymphocytes proportions and monitored body weight changes in WT C57BL/6 mice receiving NSWm7210. Our analysis revealed no significant reduction in CD3⁺ T cells in blood or spleen samples (Figure S9D). Additionally, compared to the isotype control, NSWm7210 did not induce body weight reduction in mice (Figure S9E). Collectively, these results support that NSWm7210 treatment did not induce obvious immune-related adverse events.

Construction and characterization of an anti-human CD25×TIGIT bsAb with high specificity for intratumoral Tregs

To generate an anti-human CD25×TIGIT bsAb for potential clinical application, we initially generated a series of anti-human CD25 (hCD25) mAbs by panning against a non-immune phage display human Ab library.⁴⁴ To isolate non-IL-2 blocking or partial blocking anti-CD25 mAbs, human IL-2 (hIL-2) was added to compete with the Ab library during the second round of panning, thereby non-IL-2 blocking or partial blocking phage Abs can be selected out. Subsequently, the identified anti-CD25 mAbs were screened using ELISA to identify those did not or only partially competed with IL-2 for binding to hCD25 (Figure S10A). Next, we evaluated the specific binding capabilities of these non-IL-2 blocking anti-hCD25 mAbs with flow cytometry and SPR analyses, and selected seven of them with specific binding to hCD25-expressing Raji cells and purified hCD25 protein (Figures S10B and S10C).

Using the above described seven mAbs, we constructed seven anti-hCD25×TIGIT bsAbs with the CS19 mAb, named NSWh7211–NSWh7217. To examine if anti-hCD25×TIGIT bsAbs are selective for binding to hCD25⁺ hTIGIT⁺ double-positive cells, we constructed Raji stable cell lines expressing either hCD25, hTIGIT alone, or a combination of hCD25 and hTIGIT (Figure S11A and Table S3). In flow-cytometry-based binding assays we found that, compared to a control anti-hCD25 mAb (daclizumab), our anti-hCD25×TIGIT bsAbs exhibited stronger binding to hCD25⁺ hTIGIT⁺ Raji cells while exhibiting similar (or lower) binding to hCD25⁺ Raji cells (Figure S11B). These findings support that a dual-targeting strategy confers improved binding selectivity of CD25×TIGIT bsAbs over the anti-CD25 mAb for double-positive cells.

Seeking to select out the anti-hCD25×TIGIT bsAb with the highest binding selectivity for intratumoral Tregs from the seven NSWh7211–NSWh7217 bsAbs, we compared their binding abilities to intratumoral Tregs relative to splenic Tregs, as well as intratumoral Tregs relative to intratumoral CD8⁺ T cells. Since the CD25 binding arm of these bsAbs did not cross-react with mCD25, we used CD25 humanized mice in this assay and the subsequent assays. CD25 humanized mice were inoculated with MC38 tumor cells, and established tumors and spleens were harvested and analyzed by flow cytometry to profile the Treg and CD8⁺ T cell populations. We found that NSWh7216 exhibited the best selectivity among the seven NSWh7211–NSWh7217 bsAbs. NSWh7216 bound to 78.7% of intratumoral Tregs but only to 34.5% of splenic Tregs (Δ = 44.2%) (Figure 4A); moreover, NSWh7216 only bound to 17.0% of intratumoral CD8⁺ T cells (Figure 4B). Thus, we used NSWh7216 for subsequent functional evaluations.

Construction and characterization of an anti-human CD25×TIGIT bsAb with high specificity for intratumoral Tregs

(A and B) The binding activity of anti-hCD25×TIGIT bsAbs for intratumoral Tregs and CD8⁺ T cells, and splenic Tregs. CD25 humanized mice were inoculated with 300,000 MC38 tumor cells. Tumors and spleens were taken out and analyzed once tumors reached 500–1,000 mm³ on day 18. Anti-hCD25×TIGIT bsAbs (2 μg/mL) with the human Fc DANG variant were individually incubated with tumor cells or splenocytes. Then their binding with different intratumoral Tregs and splenic Tregs, and intratumoral CD8⁺ T cells, was evaluated by FACS. The percentages of bsAb⁺ Tregs in the tumor microenvironment or spleens were compared among the indicated bsAbs (A). The percentages of bsAb⁺ Tregs or CD8⁺ T cells in the tumor microenvironment were compared among the indicated bsAbs (B). Data shown as the mean ± SEM of duplicate wells; n = 2 per group (A and B). (C) Evaluation of IL-2 blocking ability of anti-hCD25 mAbs and NSWh7216. CD3⁺ T cells isolated from human PBMCs were stimulated with 100 U/mL of hIL-2 in the presence of 1F3 mAb, NSWh7216, or control Abs. Cells were stained for pSTAT5 and the pSTAT5 signal in Tregs and Teffs and were analyzed using FACS. The representative histograms showing changes of pSTAT5 signal density observed in Tregs after incubation with the indicated Abs. (D) The binding selectivity of NSWh7216 for engineered CD25⁺ TIGIT⁺ double-positive cells. Pre-stained hCD25⁺ hTIGIT⁺ Raji cells and hCD25⁺ Raji cells were mixed with non-stained hTIGIT⁺ Raji cells at a 1:1:1 ratio. Raji cells were incubated with serially diluted NSWh7216 prior to staining with a PE-conjugated anti-hIgG secondary Ab. The binding of bsAb with different populations of Raji cells was assessed based on the PE fluorescence intensity in each cell population. The results shown are representative data for three independent experiments. (E) NSWh7216 can block the inhibitory signal derived from TIGIT-CD155 interaction in CD25⁺ TIGIT⁺ double-positive reporter cells. Two-cell luciferase reporter-based TIGIT blockade assay was used to evaluate the TIGIT blockade function of NSWh7216. CHO-OKT3 scFv-CD155 and CHO-OKT3 scFv cells were used as target cells; Jurkat-NFAT-hTIGIT and Jurkat-NFAT-hTIGIT-hCD25 were used as effector cells. Effector cells and target cells were incubated at an E:T ratio of 6:1 in the presence of the indicated Abs for 20 h. The addition of an anti-TIGIT Ab blocked the inhibitory signal mediated via TIGIT-CD155 interaction, thereby facilitating NFAT activation and luciferase gene expression. Luciferase expression was measured through the light signal produced after interacting with QUANTI-Luc assay solution. Percentages of inhibition were calculated according to the formula inhibition = 100 × ((signal of Ab group – signal of no blockade group)/signal of no blockade group). Data shown as the mean ± SEM of duplicate wells and are representative of two independent experiments; statistical significance was determined using two-way ANOVA. See also Figures S10–S13 and Table S3.

We next examined the IL-2 blocking capacity of NSWh7216 by quantification of pSTAT5. CD3⁺ T cells isolated from human peripheral blood mononuclear cells (PBMCs) were stimulated with hIL-2 in the presence of anti-hCD25 Abs, including NSWh7216, 1F3 (the anti-hCD25 mAb used for constructing NSWh7216), and two hIL-2-blocking mAbs (daclizumab and NARA1). Following IL-2 stimulation, Foxp3⁺ Tregs exhibited an approximately 69.4% increase in pSTAT5 signal intensity (Figure 4C), while CD8⁺ or CD4⁺ Teffs exhibited no increase in the pSTAT5 signal (Figures S11C and S11D). Pre-treatment of CD3⁺ T cells with daclizumab or NARA1 eliminated the pSTAT5 signal; in contrast, 1F3 mAb induced a 25.0% reduction, and NSWh7216 only induced about 21.5% reduction in the pSTAT5 signal intensity (Figure 4C). These results show that NSWh7216 only weakly inhibits IL-2 binding with CD25, suggesting it will have minimal impact on IL-2 mediated Teff function.

To compare the selectivity of NSWh7216 with its parental mAbs, cell populations expressing either both antigens (hCD25⁺ hTIGIT⁺ Raji cells) or a single antigen (hCD25⁺ Raji cells or hTIGIT⁺ Raji cells) were pre-stained with various tracer dyes, mixed at equal ratios, and incubated with serial dilutions of NSWh7216, 1F3 mAb, or CS19 mAb. Flow cytometry analysis showed that NSWh7216 exhibited much stronger binding activity for hCD25⁺ hTIGIT⁺ double-positive Raji cells than for hCD25⁺ Raji cells or hTIGIT⁺ single-positive Raji cells. This was shown by its relatively low EC₅₀ (0.26 μg/mL) for binding to the double-positive Raji cells, but its binding with both single-positive Raji cells was weak and the EC₅₀ could not be measured accurately; moreover, the maximal binding capacity for double-positive Raji cells was at least 15-fold and 3-fold higher than that for hCD25⁺ Raji cells and hTIGIT⁺ Raji cells, respectively. In contrast, 1F3 and CS19 showed no selectivity for hCD25⁺ hTIGIT⁺ Raji cells (Figure 4D). In addition, we assessed whether one NSWh7216 bsAb can simultaneously bind to two separate hCD25⁺ hTIGIT⁺ double-positive cells through CD25 and TIGIT by using a flow-cytometry-based co-binding assay (see section “materials and methods”) and found that one NSWh7216 bsAb can hardly bind to two separate hCD25⁺ hTIGIT⁺ double-positive cells at the same time (Figure S12). Thus, we have constructed an anti-hCD25×TIGIT bsAb (NSWh7216) that is selective for hCD25⁺ hTIGIT⁺ double-positive cells, most likely via one-bsAb-one-cell binding model.

One of the anti-tumor mechanisms of anti-TIGIT mAbs involves blocking the inhibitory signal transmitted from TIGIT-CD155 interaction, thereby restoring the function of Teffs.⁵²^,⁵³ To investigate whether NSWh7216 has the ability for TIGIT receptor blockade, we used a two-cell luciferase reporter assay. For the two-cell luciferase reporter assay, two engineered Jurkat cell lines (Jurkat-NFAT-hTIGIT or Jurkat-NFAT-hTIGIT-hCD25) were used as effector cells and an engineered CHO cell line (CHO-CD3 scFv-CD155) were used as target cells (Figures S13A and S13B). In the absence of Ab treatment, the TIGIT-CD155 inhibitory signal induced an approximately 40% inhibition of NFAT response element-controlled luciferase gene expression in Jurkat-NFAT-hTIGIT cells (Figure 4E). As expected, CS19 mAb elicited a significant blockade of the TIGIT-CD155 inhibitory signal in both Jurkat-NFAT-hTIGIT cells and Jurkat-NFAT-hTIGIT-hCD25 cells (Figure 4E). In contrast, NSWh7216 only elicited a significant blockade of the TIGIT-CD155 inhibitory signal in Jurkat-NFAT-hTIGIT-hCD25 cells, which was much stronger than CS19 mAb and Ctrl/CS19 bsAb (Figure 4E, right). Thus, NSWh7216 has the ability for TIGIT receptor inhibition in hCD25⁺ hTIGIT⁺ double-positive cells but not in TIGIT⁺ single-positive cells.

NSWh7216 promotes Treg depletion and tumor suppression in tumor-bearing CD25 humanized mice

Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are the major Ab Fc-mediated effector mechanisms triggering targeted cell lysis and phagocytosis.⁵⁴^,⁵⁵ To examine whether NSWh7216 exhibits selectivity in promoting ADCC and ADCP of hCD25⁺ hTIGIT⁺ double-positive cells compared to the parental mAbs, we first compared the ADCC and ADCP activities of NSWh7216, CS19, and 1F3 mAb. For the ADCC assay, a reporter system in which engineered Jurkat T cells stably expressing human FcγRIIIa receptor (a major ADCC-mediating Fc receptor⁵⁶) and an NFAT response element-driven firefly luciferase reporter was created and used as effecter cells; the target cells were hCD25⁺ hTIGIT⁺ Raji cells or hCD25⁺ Raji cells. NSWh7216 induced much stronger ADCC activity in hCD25⁺ hTIGIT⁺ Raji cells than CS19 and 1F3 mAbs; meanwhile, NSWh7216 induced stronger ADCC activity in hCD25⁺ hTIGIT⁺ Raji cells than that in hCD25⁺ Raji cells (Figure 5A).

For the ADCP assay, bone marrow-derived macrophages (BMDMs) from the C57BL/6 mice were used as effector cells; the target cells were a mixture of three types of pre-stained Raji cells at a 1:1:1 ratio. NSWh7216 induced a 17.7-fold and a 1.6-fold stronger ADCP activity in hCD25⁺ hTIGIT⁺ Raji cells than that in hCD25⁺ Raji cells and hTIGIT⁺ Raji cells, respectively (Figure 5B). In contrast, 1F3 and CS19 induced a similar level of ADCP activity in hCD25⁺ hTIGIT⁺ Raji cells as that in hCD25⁺ Raji (for 1F3) or in hTIGIT⁺ Raji cells (for CS19). Compared to NSWh7216, the combination of 1F3 and CS19 did not induce stronger ADCP activity in hCD25⁺ hTIGIT⁺ Raji cells. In addition, NSWh7216 bearing a human Fc DANG variant totally aborted its ADCP activity, confirming that NSWh7216 indeed depends on its Fc to mediate the phagocytosis (Figure 5B). These findings indicate that NSWh7216 exhibits selectivity in promoting ADCC and ADCP of hCD25⁺ hTIGIT⁺ double-positive cells compared to the parental mAbs.

We next compared the anti-tumor activity of NSWh7216 with 1F3 mAb in an MC38 tumor model using CD25 humanized mice: four doses of NSWh7216 significantly suppressed MC38 tumor growth, while 1F3 mAb did not suppress tumor growth (Figure 5C). The superior anti-tumor activity of NSWh7216 compared to its parental mAbs in vivo is consistent with its stronger ADCC and ADCP activities in vitro. A comparison between NSWh7216 and a combination therapy comprising 1F3 and CS19 mAbs showed that they conferred equal anti-tumor activity in the MC38 tumor model (Figure 5D).

It has been reported that afucosylation of IgG-Fc can enhance anti-tumor activity by promoting the interaction of IgG with FcγRIII.⁵⁷^,⁵⁸ Seeking to further enhance the Fc-mediated functions of NSWh7216, we used an engineered HEK293F cell line (wherein the FUT8 gene is inactivated⁵⁹) to express afucosylated NSWh7216 (NSWh7216-afu). When comparing the anti-tumor activity of NSWh7216 with NSWh7216-afu in an MC38 tumor model using CD25 humanized mice, we found that NSWh7216-afu conferred enhanced tumor suppression (Figure S14).

After confirming that the NSWh7216 monotherapy confers an anti-tumor effect, we examined whether NSWh7216 can synergize with anti-mPD-1 Ab in the MC38 tumor model, specifically by comparing the anti-tumor efficacy of anti-mPD-1 monotherapy, a combination therapy comprising NSWh7216 and anti-mPD-1, and a combination therapy comprising 1F3 and anti-mPD-1. Once tumors reached 80–200 mm³, the tumor-bearing mice were divided and treated with two doses of NSWh7216 or 1F3 (10 mg/kg), followed by anti-mPD-1 mAbs (3 mg/kg) administration twice a week (Figure S15A). Compared to the vehicle control, the anti-mPD-1 monotherapy did not effectively inhibit MC38 tumor growth. However, the combination therapy of NSWh7216 and anti-mPD-1 outperformed the combination therapy of 1F3 and anti-mPD-1 in inhibiting MC38 tumor growth (Figure S15B). We also observed that, compared to the anti-mPD-1, the NSWh7216 and anti-mPD-1 combination therapy significantly prolonged the survival of MC38 tumor-bearing mice, doing so without inducing body weight reduction (Figures S15C and S15D). Taken together, these results indicate that NSWh7216 can synergize with another immune checkpoint inhibitor (e.g., anti-PD-1 Abs) to improve anti-tumor efficacy.

After confirming the anti-tumor activity of NSWh7216 in vivo, we examined whether NSWh7216 selectively leads to decreases in the proportion of intratumoral Tregs but not in normal tissues, specifically by assessing Treg depletion in tumor, spleens, and peripheral blood after one dose of NSWh7216 treatment in CD25 humanized mice bearing MC38 tumors. Compared to the isotype mAb, NSWh7216 led to a 30% decrease of the percentage of intratumoral Tregs without depleting Tregs in spleens or peripheral blood after one dose of Ab treatment (Figure 5E). Additionally, we did not observe decreases in the percentages of CD8⁺ T cells or CD4⁺ T cells in the tumor, blood, or spleens after a single dose of NSWh7216 treatment (Figure S16). Taken together, these results demonstrate that NSWh7216 treatment can suppress tumor growth by selectively depleting intratumoral Tregs while avoiding non-selectively depleting peripheral Tregs.

Discussion

Lack of a marker specific for Tregs is a major obstacle limiting the utility of Ab-based Treg depletion therapies in clinical cancer treatment.⁶⁰ Considerable research efforts have been undertaken to enhance the target-binding specificity of Abs for immune modulatory receptors, employing a myriad of bispecific Ab formats.⁶¹^,⁶²^,⁶³ We have here investigated the CD25×TIGIT bsAb in cancer therapy in terms of efficacy and potential influence of peripheral Tregs. We initially constructed an anti-mCD25×TIGIT bsAb, NSWm7210. After confirming its binding selectivity with CD25⁺ TIGIT⁺ double-positive cells in vitro, we demonstrated that NSWm7210 has stronger intratumoral Treg depletion, Teff activation, and anti-tumor activity compared to the anti-CD25 mAb. To facilitate the potential clinical application of CD25×TIGIT bsAb, we developed an anti-hCD25×TIGIT bsAb, NSWh7216, with stronger anti-tumor activity than the parental mAbs. Notably, NSWh7216 did not significantly reduce peripheral Tregs, indicating the CD25×TIGIT bsAb is an effective strategy for selective intratumoral Treg depletion with minimal toxic effects on peripheral Tregs.

Previous studies have adapted the bsAb strategy to selectively target immune cells within the tumor microenvironment, thereby activating their anti-tumor activity through modulating specific immune modulatory receptors.⁶⁴ For example, based on the co-expression of CTLA-4 and PD-1 on TILs but not on peripheral T cells, various formats of PD-1×CTLA-4 bsAbs have been investigated to activate CTLA-4⁺ PD-1⁺ double-positive TILs.⁶¹^,⁶²^,⁶⁵ We found murine TILs, particularly intratumoral Tregs, co-express CD25 and TIGIT at much higher levels compared to Tregs derived from spleens and peripheral blood. Consistently, by analyzing single-cell RNA sequencing (scRNA-seq) data from 21 tumor types,⁴¹ we also found that CD25 and TIGIT were co-expressed at higher levels on human intratumoral Tregs. The co-expression pattern of CD25 and TIGIT on intratumoral Tregs supports that a dual-targeting strategy can serve as an effective approach to enhance the targeting selectivity of intratumoral Tregs.

Immunotherapies based on anti-CD25 mAbs before tumor establishment have been demonstrated as effective for prophylaxis for mouse tumor growth⁶⁶; however, administration of anti-CD25 mAbs against established tumors failed to delay tumor growth.⁶⁷ A recently developed non-IL-2 blocking anti-CD25 mAb (RG6292)²⁴ is under clinical development, and results in patients with solid tumors have indicated that RG6292 is well tolerated (without a fatal outcome), both as a single agent and as a combination agent with atezolizumab.⁶⁸ Regarding clinical progress with anti-TIGIT Abs, they have shown little activity when used as monotherapies in advanced solid tumors.⁶⁹ Nevertheless, several of their combinations with additional mAbs (such anti-PD-1 and PD-L1 mAbs) showed therapeutic benefit in patients with non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC).⁴⁰^,⁷⁰ Thus, developing efficient therapeutic Abs that target intratumoral Tregs remains highly desirable.

In our study, we found that NSWh7216 has a much stronger binding avidity with CD25⁺ TIGIT⁺ double-positive cells compared to its binding with CD25⁺ single-positive cells, which is shown by an at least 15-fold higher maximal binding capacity for double-positive cells than for CD25⁺ single-positive cells. This finding is consistent with previous studies that have highlighted the capacity of bsAb to improve target selectivity by cross-arm avidity binding to two antigens present on the surface of the same cells.⁷¹^,⁷² Based on the co-expression of CD25 and TIGIT on intratumoral Tregs—but not on peripheral Tregs—NSWh7216 indeed showed binding selectivity for intratumoral Tregs in vivo, and it can contribute to suppression of established tumors. Systemic Treg depletion leads to severe autoimmune toxicity in mouse models (e.g., the Foxp3-DTR mouse model⁵¹^,⁷³), whereas NSWh7216 did not reduce the percentage of peripheral Tregs, supporting that NSWh7216 holds promise to alleviate these safety concerns.

Notably, unlike NSWm7210 and other anti-hCD25×TIGIT bsAbs that we screened in this study—which all apparently exerted stronger influences on peripheral Tregs than NSWh7216—NSWh7216 was generated by combining a low-affinity anti-CD25 mAb with the same anti-TIGIT mAb CS19. Therefore NSWh7216’s relatively low-affinity arm for binding to CD25 may partially contribute to its better binding selectivity. Moreover, other forms of bsAb, such as monovalent DART,⁷⁴ which use monovalent scFv instead of a bivalent Fab region, may help increase the specificity of intratumoral Tregs targeting by reducing the binding avidity of CD25×TIGIT bsAbs to Teffs when a relatively high-affinity anti-CD25 Ab is chosen as one arm of CD25×TIGIT bsAbs.

It is also worth noting that, despite our finding that CD25⁺TIGIT⁺ double-positive cells comprise about 30% of the intratumoral CD4⁺ Foxp3⁻ Teffs in the MC38 tumor model, NSWm7210 treatment did not lead to significant intratumoral CD4⁺ Foxp3⁻ Teffs decrease. This can be attributed to the lower expression levels of CD25 and TIGIT on the intratumoral CD4⁺ Foxp3⁻ Teffs compared to their expression on Tregs.

Previous studies have provided evidence that both an Fc-mediated depletive capacity and a Fab-mediated blocking capacity are necessary for controlling the overall activity of immunomodulatory Abs.⁷⁵^,⁷⁶^,⁷⁷ We also found that CD25×TIGIT bsAbs combine an Fc-mediated depletive capacity and a Fab-mediated blocking capacity in the same molecule. All of the bsAbs used in this investigation are of the human IgG1 (hIgG1) isotype; hIgG1 has a high FcγRs-binding affinity and primarily acts via ADCC and ADCP to confer therapeutic efficacy.⁷⁸ Although we did not formally assess which immune cells are the mediators of Treg depletion, our NK cell-mediated in vitro cytotoxicity assay data suggests that NK cells should be able to drive Treg depletion. Anti-CD25 mAbs such as PC61 and RG6292 have been demonstrated to induce Treg depletion in a macrophage-dependent manner.²⁴^,⁷⁹ Our macrophage-mediated in vitro phagocytosis assay data suggests that macrophages may also play a role in CD25×TIGIT bsAbs-mediated Treg depletion.

Considering that the duration of Treg depletion is relatively short after a single dose of NSWm7210 administration, more frequent administration of NSWm7210 may be required to maintain intratumoral Tregs at a therapeutically relevant level of depletion. Although Treg rebound may limit the long-term effectiveness of this approach, note that the activation of effector cells has already occurred prior to Treg rebound, and a combination therapy with other immune checkpoint inhibitors may further enhance the function of these effector cells. In addition to CD25 and TIGIT, other markers with relatively high expression on Tregs and low expression on Teffs could be explored as potential targets (for example, CTLA-4 and CCR8). Notably, CD25×CTLA-4 bsAbs¹³^,¹⁴ and CCR8×CTLA-4 bsAbs⁸⁰^,⁸¹ have been reported to achieve selective depletion of intratumoral Tregs.

In conclusion, leveraging the co-expression of CD25 and TIGIT on intratumoral Tregs but not on peripheral Tregs, we have developed an anti-hCD25×TIGIT bsAb and demonstrated its effectiveness and selectivity for targeting intratumoral Tregs to suppress established tumors. Considering that Treg depletion strategies in combination with immune checkpoint inhibitors (ICIs) (such as anti-PD-1 and PD-L1) have shown promise for overcoming resistance to monotherapies of immune checkpoint receptors,²¹^,⁸² our CD25×TIGIT bsAb can be viewed as a potential Treg depletion strategy to enhance the therapeutic efficacy of ICIs.

Materials and methods

The scRNA-seq data analysis

Firstly, the per-cell size-factor normalization and per-gene Z score scaling across cells for each dataset were performed. Then, cells within each dataset were partitioned into small groups (miniclusters) to reduce noise. Subsequently, a batch-effect correction algorithm, Harmony, was applied to further improve the integration. On the basis of the Harmony result, Seurat was applied to identify clusters, termed metaclusters. For a more detailed description of the data processing steps, please refer to the previously published work.⁴¹

Cell lines

Raji and CHO cell lines were from the Cell Bank of Type Culture Collection, Chinese Academy of Sciences, or American Type Culture Collection. The hCD25⁺ Raji, mCD25⁺ CHO cell lines were established by stably expressing full length of human CD25 (hCD25) or mCD25. hTIGIT⁺ Raji, mTIGIT⁺ CHO cell lines were established by stably expressing full length of hTIGIT or mouse TIGIT (mTIGIT). hCD25⁺hTIGIT⁺ Raji cell line was constructed by stably expressing full length of hCD25 on hTIGIT⁺ Raji cell line and the mCD25⁺ mTIGIT⁺ CHO cell line was constructed by stably expressing full length of mTIGIT on mCD25⁺ CHO cell line. MC38 and the HEK293F were from Life Technologies. The E.G7 cell line (a derivative of EL4 that expresses OVA) was provided by Dr. Chen (NIBS). The CHO-OKT3 scFv-CD155 cell line and the Jurkat-NFAT-hTIGIT cell line were provided by Huahui Health. The Jurkat-NFAT-hTIGIT-hCD25 was established by stably expressing full-length hCD25 on the Jurkat-NFAT-hTIGIT cell line.

BsAb construction

The variable heavy-chain (VH) and variable light-chain (VL) gene sequences of 7D4, PC61, daclizumab, and NARA1 were synthesized by Shanghai Generay Biotech. CS19 and series of anti-hCD25 were selected from our human non-immune scFv phage display Ab library.⁴⁴ To construct the bsAbs, the original WT HC expression vectors (blank) were modified by Dr. Du (NIBS) by introducing several mutations into the constant heavy chain 3 (CH3) of the heavy chain (HC) via site-directed mutagenesis to generate the knob chain (T366W, S354C) and the hole chain (Y349C, T366S, L368A, Y407V) according to the knob-into-hole technology. In order to increase the chance of correct pairing of the specific VH and its VL, the CH1 of the hole chain and the constant light chain (CL) of the light chain (LC) were interchanged to generate the paired crossover HC (CL-hole chain) and LC (CH1-LC) according to Cross-Mab technology. The VH and VL fragments of CS19 were cloned into CL-hole chain and CH1-LC chain expression vectors, respectively. 7D4 and series of anti-hCD25 mAbs were cloned into the knob chain.

Expression and purification of proteins

The extracellular domains (ECDs) of CD25, TIGIT, and CD155 were produced as His 6-, His 6 -Avi–, or Fc-tagged fusion proteins by transient transfection of HEK293F cells and were purified by affinity chromatography. For full-length IgG Abs, the coding sequences of the V regions of HC and LC were subcloned into human IgG1 HC expression vector and LC expression vector, respectively. 293F cells were co-transfected with the two IgG expression plasmids (HC + LC plasmids) at a 1:1 ratio. For bsAbs expression, 293F cells were co-transfected with the four IgG expression plasmids (HC knob+ LC WT +HC hole + LC hole plasmids) at a 1:1:1:1ratio. After 4–6 days of transfection, the 293F cells supernatants were collected for purification of IgG1 via protein A beads affinity chromatography.

For afucosylated Abs expression, HEK293F cells (293F Fut8^−/−) with an inactivated FUT8 gene were co-transfected with the plasmids encoding the corresponding H chain and L chain. After 4–6 days, the HEK293F cell supernatants were collected for purification of IgG1 via protein A bead affinity chromatography.

Ab library panning and screening of anti-human CD25 Abs

For anti-human CD25 Ab panning, the ECD of hCD25 was fused with His₆ -Avi tag and biotinylated by BirA ligase. Biotinylated hCD25 ECD was used as antigen in the panning experiments with a non-immune human Ab library.⁴⁴ Phage-scFvs were screened after two rounds of selection for specific binding with biotinylated hCD25 ECD. During the second round of panning, a 300-fold higher molar concentration of human IL-2 (hIL-2) relative to biotinylated hCD25 ECD was added. This addition was made to create a competitive environment where the excess hIL-2 competed with the Ab library for binding to CD25, for non-IL-2 blocking or partial blocking phage Abs can be selected out. After two rounds of panning, a total of about 600 single clones were randomly picked and screened for binding to hCD25 by ELISA. Clones selected out were produced as purified phage-scFv particles or converted into the full-length human IgG1 format. Subsequently, the output anti-hCD25 mAbs were screened using ELISA to identify those that do not or only partially compete with IL-2 for binding to hCD25.

ELISA-based binding and competition assays

For the ELISA-based binding assay, biotinylated CD25 ECD was captured with streptavidin (Sigma-Aldrich)-coated 96-well plates (MaxiSorp; Nunc). For phage-scFv-based ELISA, serially diluted phage-scFvs were added and then detected by adding mouse anti-M13-horseradish peroxidase (HRP) Ab (GE Healthcare). For full-length human IgG-based ELISA, coating the antigen and diluting mAbs with a similar method to the phage-scFvs. Then the bound Abs were detected using a mouse anti-human IgG-Fc-HRP Ab (Thermo Fisher Scientific). For the ELISA-based competition assays for anti-hCD25 mAbs, hCD25 ECD was coated on 96-well plates overnight at 4°C, then anti-hCD25 mAbs at serially diluted concentrations were mixed with 1.5 μg/mL of biotinylated hIL-2 and added to the ELISA plates to compete for the binding between hIL-2 and hCD25. The signal was measured via ligand detection using HRP-anti-streptavidin secondary Ab (Pierce).

For the ELISA-based competition assays for CS19 mAb, the biotinylated antigens hTIGIT ECD or mTIGIT ECD were coated with a similar method to the phage-scFvs. CS19 hIgG1 at serially diluted concentrations were mixed with 2 μg/mL of hCD155-mFc or mCD155-mFc and added to the ELISA plates to compete for the binding between TIGIT and CD155. The signal was measured via ligand detection using HRP-anti-mouse IgG secondary Ab (Thermo Fisher Scientific).

In vivo evaluation of ADAs

The development of ADAs was monitored in WT C57BL/6 mice after NSWm7210 treatment. Ten milligrams per kilogram of NSWm7210 was given twice per week five times. Blood serum samples were collected at baseline (day 0), as well as on days 3, 7, 14, 21, and 28 prior to each NSWm7210 administration. The ADAs in serum samples were measured using ELISA. For the ELISA-based ADA measurement, NSWm7210 was directly coated on 96-well plates (MaxiSorp; Nunc) overnight at 4°C. Then the collected serum samples were diluted (1:20) and added to the ELISA plates. An HRP-conjugated goat anti-mouse IgG Ab (minimal x-reactivity, BioLegend) was used to detect anti-NSWm7210’s ADAs.

PK analysis in mice

A single-dose PK study was carried out in WT C57BL/6 mice, in which 8- to 10-week-old female C57BL/6 mice were injected i.p. with 10 mg/kg of each Ab. Blood samples were collected at different time points, and serum concentrations of human Ab were measured with an hIgG ELISA quantitation kit (Bethyl Laboratories). Then Ab serum concentration was calculated and plotted using Prism 6. The evaluation of the PK data was conducted using WinNonlin.

SPR analysis

Kinetic analysis of the bindings of anti-CD25, anti-TIGIT mAbs to the corresponding antigens were performed on a Biacore T200 (Biacore, GE Healthcare). Anti-hFc Ab (Thermo Fisher) was covalently attached to the surface of a CM5 sensor chip using an amine coupling kit (GE Healthcare). Abs at optimal concentrations were captured on the chip and the analytes (CD25 or TIGIT) were then injected at 2-fold serial diluted concentrations. The association rates (Kon), dissociation rates (Koff), and affinity constants (K_D) were calculated using BiacoreT200 evaluation software.

For simultaneous binding analysis of NSWm7210 to the corresponding antigens, mCD25 was firstly covalently attached to a CM5 sensor chip using an amine coupling kit (Biacore), then NSWm7210 was injected at 3 μg/mL with a dissociation phase of 30 s, and mTIGIT was injected with a dissociation phase of 60 s, followed by regeneration with pH3.0 Glycin-Hcl.

Flow-cytometry-based binding assays

To compare binding abilities of anti-hCD25 mAbs and anti-human CD25×TIGIT bsAbs to hCD25⁺ Raji cells or hCD25⁺ hTIGIT⁺ Raji cells, cells were prepared and incubated with diluted Abs for 30 min at 4°C in 1% BSA/PBS, then these cells were washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-hIgG secondary Ab (Sigma-Aldrich) for 20 min at 4°C. These samples were analyzed with a FACSAria II instrument (BD Biosciences), and data were processed using FlowJo version (v.) 10 software.

To test the binding selectivity of CD25×TIGIT bsAbs, CD25⁺TIGIT⁺ CHO/Raji cells were labeled with 0.2 μM CellTrace Violet dyes, and CD25⁺ CHO/Raji cells were labeled with 0.5 μM CellTrace Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) according to the manufacturer’s protocol (Invitrogen) followed by mixing two labeled cells with unlabeled TIGIT⁺ CHO/Raji cells in an equal cell ratio. The mixed cells were incubated with serially diluted Abs for 30 min at 4°C, then these cells were washed and incubated with PE-conjugated goat anti-hIgG secondary Ab (Sigma-Aldrich) for 20 min at 4°C. These samples were analyzed with a FACSAria III instrument (BD Biosciences), and data were processed using FlowJo version (v.) 10 software.

Flow-cytometry-based co-binding assay

To assess whether NSWh7216 can bind to two separate hCD25⁺ hTIGIT⁺ double-positive cells through CD25 and TIGIT, we have designed a flow-cytometry-based co-binding assay. The co-binding assay was conducted as follows: first, the double-positive cells were pre-stained with two different dyes separately, and 2 μM CellTrace Violet dyes and 1 μM CellTrace CFSE were used for staining according to the manufacturer’s protocol (Invitrogen). Then, the pre-stained cells were mixed (1:1) and incubated with diluted Abs (including NSWh7216, 1F3/Ctrl bsAb, Ctrl/CS19 bsAb, and 1F3 mAb) for 30 min at 4°C. If NSWh7216 can co-bind to two double-positive cells through CD25 and TIGIT, then it would bridge the CFSE⁺ and Violet⁺ cells, resulting in a CFSE⁺ Violet⁺ double-positive population. Finally, this co-binding was measured using flow cytometry (indicated as the percentage of cells in the upper right quadrant of a CFSE vs. Violet scatterplot), representing the CFSE⁺ Violet⁺ double-positive population. These samples were analyzed with a FACSAria III instrument (BD Biosciences), and data were processed using FlowJo version (v.) 10 software.

ADCC assay

For ADCC assay, a Jurkat cell line stably expressing human FcγRIIIa (hFcγRIIIa) receptor and an NFAT response element-driven firefly luciferase reporter (Jurkat-NFAT-hFcγRIIIa) was created and used as effecter cells. hCD25⁺ hTIGIT⁺ Raji cells and hCD25⁺ Raji cells were used as target cells. Target cells (15,000 per well) were seeded into wells of a 96-well solid white polystyrene microplate (Corning) and incubated with 3-fold serially diluted NSWh7216, 1F3, CS19, and isotype Abs. Jurkat-NFAT-hFcγRIIIa effector cells (75,000 per well) were then added into wells containing the target cells and Abs and incubated for 6 h at 37°C. ADCC activity was determined by measuring luciferase expression according to the instruction of Bright-Glo Luciferase Assay reagents (Promega).

ADCP assay

For the ADCP assay, BMDMs from C57BL/6 mice were used as effector cells. To prepare BMDMs, mouse bone marrow cells were collected from the tibias and femurs of C57BL/6 mice, and the cells were subsequently induced by granulocyte-macrophage colony-stimulating factor in L929 supernatants for 7 days. The differentiated BMDMs (200,000 cells/well of a 24-well plate) were prepared 1 day before analysis. hCD25⁺ hTIGIT⁺ Raji cells, hCD25⁺ Raji cells, and hTIGIT⁺ Raji cells were used as target cells after they were stained with 5 μM CellTrace CFSE, 5 μM CellTrace Violet, and 5 μM CellTrace Yellow dyes according to the manufacturer’s protocol (Invitrogen). The BMDMs were labeled with anti-mouse F4/80-Alexa Fluor 647 (Thermo Scientific) prior to incubation with target cells. The mixture of target cells was incubated with different Abs at room temperature for 15 min and then added to the labeled BMDMs using an effector-to-target ratio of about 1:3 for 2 h at 37°C. Phagocytosis of three dye-labeled target cells by anti-mouse F4/80 Ab-labeled macrophages was recorded using a Nikon A1+SIM confocal microscope.

Animal studies

All animal experiments were conducted following the National Guidelines for Housing and Care of Laboratory Animals in China and performed under the approved Institutional Animal Care and Use Committee protocols at the National Institute of Biological Sciences, Beijing, China.

For mouse tumor models, 6- to 8-week-old female C57BL/6 mice or CD25 humanized C57BL/6 mice were inoculated subcutaneously (s.c.) with 300,000 MC38, 500,000 E.G7 cells, and 300,000 MCA205 cells in the right flank; 6- to 8-week-old female BALB/c mice were inoculated s.c. with 200,000 CT26 tumor cells in the right flank. When tumor volumes reached 80–200 mm³ for MC38, E.G7, and MCA205 tumors, or 100–400 mm³ for CT26 tumors, mice were randomly divided into five groups and received an i.p. injection of Abs twice per week for multiple injections. Tumor volume was measured by caliper and calculated using the modified ellipsoid equation 1/2 × (length × width²). Both C57BL/6 and BALB/c mice were purchased from Charles River, and CD25 humanized C57BL/6 mice were purchased form Shanghai Model Organisms Center.

pSTAT5 assay

Mouse CD3⁺ T cells were sorted by FACS from splenocytes (after red blood cell [RBC] lysis). Human CD3⁺ T cells were isolated from PBMC using EasySep Human T cell Enrichment Kit (STEMCELL, catalog no. 19051), and 300,000 mouse CD3⁺ T cells or human CD3⁺ T cells in complete RPMI culture medium were plated and rested for 1 h at 37°C. Abs were then added at 10 μg/mL and incubated with cells for 30 min at 37°C (7D4, PC61, NARA1 mAbs, or NSWm7210 were used for pSTAT5 staining in mouse CD3⁺ T cells; 1F3, daclizumab, NARA1 mAbs, or NSWh7216 bsAbs were used for pSTAT5 staining in human CD3⁺ T cells), following which cells were stimulated with 100 IU human IL-2 for 30 min at 37°C. Before pSTAT5 and other makers’ staining, cells were stained with LIVE/DEAD Fixable Near-IR Stain Kit (Thermo Fisher Scientific) to exclude the dead cells, then they were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and BD Phosflow Perm Buffer III. The fluorescence-conjugated Abs used were as follows: pSTAT5 Pacific Blue (clone 47/Stat5(pY694), BD Biosciences), anti-mCD4-FITC (clone GK1.5, BioLegend), anti-mCD8-percp-Cy5.5 (clone 53–6.7, BioLegend), and anti-mFoxp3-APC (clone FJK-16s; eBioscience) for mouse T cells staining; pSTAT5 Pacific Blue (clone 47/Stat5(pY694), BD Biosciences), anti-hCD4-PerCp-ef710 (clone SK3, eBioscience), anti-hCD8-FITC (clone HIT8a, BD Biosciences), anti-hFoxp3-ef660 (clone 236 A/E7, eBioscience), and anti-hCD45RA-BV605(clone HI100, BioLegend). After Abs staining, these samples were analyzed with a FACS Fusion instrument (BD Biosciences), and the obtained data were processed using FlowJo version (v.) 10 software. Percentages of blockade were calculated according to the following equation: blocking = 100 × ((% pSTAT5⁺ cells of no-Ab group − % pSTAT5⁺ cells of Ab group)/(% pSTAT5⁺ cells of no-Ab group)).

Tissue processing and flow cytometry analyses of immune cells

Blood, spleen, and tumors from mice were harvested, and single-cell suspensions were prepared for FACS analyses. Blood was collected from submandibular vein, and both blood and the dissociated spleens were treated with RBC lysis buffer. MC38 tumors were dissected into small pieces and dissociated according to Tumor Dissociation Kit (Miltenyi Biotec, order no. 130-096-730), CT26 tumors were dissected into small pieces and digested with dissociation solution (PBS, collagenase type I [200 U/mL], and DNase I [60 mg/mL]) for 30 min at 37°C in a shaker at 180 rpm. E.G7 and MCA205 tumors were dissected without enzyme dissociation after dissociation cells were passed through a 40-μm cell strainer to obtain single-cell suspensions.

For surface marker staining, cells were incubated with anti-mouse FcγRⅡ/FcγRⅢ Ab (clone 2.4G2, BD Biosciences) to block FcγRs binding for 15 min. Then cells were staining with the following fluorescence-conjugated Abs: anti-mCD45-FITC (clone 30F11, BioLegend), an mCD3-PE (clone 17A2, BioLegend), anti-mCD8-percp-Cy5.5(clone 53–6.7, BioLegend), anti-mCD4-BV605 (clone GK1.5, BioLegend), anti-mNK1.1-BV421(clone PK136, BioLegend), anti-mCD25-AF647 (clone M-A251, BioLegend), anti-mTIGIT-BV421(clone 1G9, BioLegend), anti-mCD39-APC (clone Duha59, BioLegend), and anti-mPD-1-BV605 (clone 29F.1A12, BioLegend). A LIVE/DEAD Fixable Near-IR Stain Kit (Thermo Fisher Scientific) was used to differentiate live and dead cells. For Foxp3 transcription factor staining, cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience) and then stained with anti-Foxp3-PE/APC (clone FJK-16s; eBioscience).

For assessing the level of cytokine production using intracellular cytokine staining, single-cell suspensions were isolated from mice 3 days after one dose of Abs treatment in the MC38 tumor model. The isolated cells were activated for 3 h with a cell activation mixture containing brefeldin A (BioLegend). Activated cells were harvested for surface and intracellular cytokine staining. Anti-mCD45-FITC (clone 30F11, BioLegend), anti-mCD8-APC (clone 53-6.7, BioLegend), anti-mCD4-BV605 (clone GK1.5, BioLegend), and anti-mNK1.1-PE-Cy7(clone PK136, BioLegend) were used to identify the cellular origin of cytokines (i.e., CD8 ⁺ T cell or NK cell origin). After surface staining, the cells were fixed and permeabilized with a Cytofix/Cytoperm Kit (BD Biosciences). Intracellular cytokine staining was performed by incubating with anti-mouse IFN-γ-PE (clone XMG1.2; BioLegend) or anti-mPerforin-PE (clone S16009B; BioLegend). Samples were analyzed with a FACS Fusion instrument (BD Biosciences), and obtained data were processed using FlowJo version (v.) 10 software.

Intestines processing, flow cytometry, and histological analyses

To isolate lamina propria cells for flow cytometry analysis, the intestines from WT C57BL/6 mice were removed and the distal two-thirds of the cecum were collected. The cecum was cleaned with wash buffer, cut into small pieces, and then digested with 10 μg/mL Dispase (Worthington #LS02104), 100 μg/mL DNase (Sigma #DN25-1G), and 1 mg/mL Collagenase D (Roche #11088874103) at 37°C for 1 h. Samples were passed through a 70-μm cell strainer, pelleted, and resuspended in 3 mL of a 40% Percoll solution (GE Healthcare). The 40% Percoll solution was then carefully underlaid with 3 mL of 80% Percoll solution. After centrifugation at 2,200 rpm for 20 min with no brake, immune cells were recovered from the 40%–80% Percoll interphase. For surface marker staining, cells were incubated with anti-mouse FcγRⅡ/FcγRⅢ Ab (clone 2.4G2, BD Biosciences) to block FcγRs binding for 15 min. Then cells were stained with the following fluorescence-conjugated Abs: anti-mCD8-percp-Cy5.5 (clone 53–6.7, BioLegend), anti-mCD4-BV605 (clone GK1.5, BioLegend), anti-mCD25-AF647 (clone M-A251, BioLegend), and anti-mTIGIT-BV421(clone 1G9, BioLegend). A LIVE/DEAD Fixable Near-IR Stain Kit (Thermo Fisher Scientific) was used to differentiate live and dead cells. For Foxp3 transcription factor staining, cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience) and then stained with anti-mFoxp3-PE (clone FJK-16s; eBioscience), anti-mFoxp3-APC (clone FJK-16s; eBioscience), anti-mTbet (clone 4B10, BioLegend), and anti-mRoRγt (catalog no. 563282, BD Horizon). Meanwhile, the remaining cecum and entire large intestine were prepared as Swiss Rolls, fixed in 4% paraformaldehyde (PFA), and then processed for H&E staining.

Two-cell luciferase reporter-based TIGIT blockade assay

To test the blockade function of NSWh7216, Jurkat-NFAT-hTIGIT and Jurkat-NFAT-hTIGIT-hCD25 cells were used as two effector cells. They were constructed by stably expressing hTIGIT or a combination of hTIGIT and hCD25 into Jurkat-Lucia NFAT cells (InvivoGen), which express an NFAT response element-driven Lucia luciferase reporter. CHO-OKT3 scFv-CD155 and CHO-OKT3 scFv cells were used as target cells; they were constructed by stably expressing CD155 or anti-CD3(OKT3), and 20,000 target cells were seeded into each well of a 96-well flat plate in 100 μL of the culture medium (DMEM with 10% FBS), followed by incubation at 37°C for 2 h. Then, the culture medium was removed and effector cells (E:T = 5:1) were added into the wells in 70 μL of assay medium (Iscove's Modified Dulbecco's Medium (IMDM) with 10% FBS) per well. Next, the serially diluted Abs were added and incubated for 18–24 h at 37 °C in 35 μL of assay medium per well. Notably, in the no-blockade group, CHO-OKT3 scFv cells were incubated with effector cells and various Abs. After incubation, the plate was centrifuged at 250 × g for 4 min, then 50 μL of sample per well was pipetted into a 96-well white plate and 50 μL of QUANTI-Luc assay solution was added to each well. Percentages of inhibition were calculated according to the formula inhibition = 100 × ((signal of Ab group – signal of no blockade group)/signal of no blockade group).

Receptor density analysis

To analyze the expression of CD25 and TIGIT on CHO and Raji stable cell lines, purified 7D4-DANG, daclizumab-DANG, and CS19-DANG mAbs were biotinylated with EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific) after purification. Cells were prepared and incubated with biotinylated mAbs (10 μg/mL) for 30 min at 4°C in 1% BSA/PBS, then these cells were washed and incubated with 0.5 μg/mL PE-conjugated anti-streptavidin secondary Ab (Sigma-Aldrich) for 20 min at 4°C. These samples were analyzed with a FACS Fusion instrument (BD Biosciences), and data were processed using FlowJo version (v.) 10 software. Receptor densities were determined by BD Quantibrite Beads-PE Fluorescence Quantitation Kit. Receptor density analysis of CD25 and TIGIT expression on MC38 tumor-derived lymphocytes followed this method.

Statistical analysis

Prism 6 (GraphPad) was used for specific comparisons throughout the manuscript with p values indicated in figures. Two-way ANOVA was used to determine statistically significant differences in inhibition of tumor growth between groups in animal studies, and two-tailed unpaired Student’s t tests were used to compare two groups in the other experiments. p values < 0.05 were regarded as statistically significant.

Data and code availability

The main data supporting the results in this study are available within the paper and its supplemental information.

Acknowledgments

We thank Drs. M. Xu and Y. Miao at NIBS for their technical assistance in analyzing the intestinal tissue samples. We also thank the NIBS Animal Facility for its help in animal handling and care. This work was supported by grants from the Beijing Municipal Science and Technology Commission, and Beijing Key Laboratory of Pathogen Invasion and Immune Defense (Z171100002217064 to J.S.).

Author contributions

X.W. and J.S. conceptualized this study, interpreted the results, and drafted the manuscript. X.W., L.Z., F.Y., Y.Y., Y.L., R.F., G.S., K.W., and Z.W. performed experiments. K.D. constructed the expressing vectors of the knob chain and hole chain. H.Z. completed the scRNA-seq data analysis. X.W. prepared the figures. J.S. supervised the study. All authors commented on the manuscript.

Declaration of interests

The authors declare no competing interests.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2024.09.010.

Supplemental information

Document S1. Figures S1–S16 and Tables S1–S3

mmc1.pdf^{(12.4MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(15.6MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S16 and Tables S1–S3

mmc1.pdf^{(12.4MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(15.6MB, pdf)}

Data Availability Statement

The main data supporting the results in this study are available within the paper and its supplemental information.

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PERMALINK

A CD25×TIGIT bispecific antibody induces anti-tumor activity through selective intratumoral Treg cell depletion

Xin Wei

Linlin Zhao

Fang Yang

Yajing Yang

Huixiang Zhang

Kaixin Du

Xinxin Tian

Ruihua Fan

Guangxu Si

Kailun Wang

Yulu Li

Zhizhong Wei

Miaomiao He

Jianhua Sui

Abstract

Graphical abstract

Introduction

Results

CD25 and TIGIT are co-expressed in both murine and human intratumoral Tregs

Figure 1.

Design and characterization of a bispecific antibody targeting mouse CD25 and TIGIT

NSWm7210 suppresses the growth of established tumors in an Fc-dependent manner

Figure 2.

NSWm7210 promotes intratumoral Treg depletion and effector cell activation in the tumor microenvironment

Figure 3.

Assessment of NSWm7210’s safety profile

Construction and characterization of an anti-human CD25×TIGIT bsAb with high specificity for intratumoral Tregs

Figure 4.

NSWh7216 promotes Treg depletion and tumor suppression in tumor-bearing CD25 humanized mice

Figure 5.

Discussion

Materials and methods

The scRNA-seq data analysis

Cell lines

BsAb construction

Expression and purification of proteins

Ab library panning and screening of anti-human CD25 Abs

ELISA-based binding and competition assays

In vivo evaluation of ADAs

PK analysis in mice

SPR analysis

Flow-cytometry-based binding assays

Flow-cytometry-based co-binding assay

ADCC assay

ADCP assay

Animal studies

pSTAT5 assay

Tissue processing and flow cytometry analyses of immune cells

Intestines processing, flow cytometry, and histological analyses

Two-cell luciferase reporter-based TIGIT blockade assay

Receptor density analysis

Statistical analysis

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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